Method of preparing hemating standardized reagent



Dec. 29,- 1953 'r. E. WEICHSELBAUM METHOD OF PREPARING HEMATINSTANDARDIZED REAGENT Filed June 25, 1951 FIG. 2

FIG.

INVENTOR WEICHSELBAUM THEODORE E.

FIG. 3

ATTORNEY Patented Dec. 29, 1953 UNITED ST PARING HEMATIN METHOD OF PRESTANDARDI ZED REAGENT Continuation 'of October 8, 1947. Serial No.233,465

application Serial No. 778,691, This application June 25, 1951,

2 Claims. (Cl. 252-408) This invention relates to certain new and usefulimprovements in methods of preparing quantitative laboratory standardsof the type used in clinical chemistry for verifying and correctinginstrument calibration tables and graphs. This application is acontinuation of my co-pending application Serial No. 778,691, filedOctober 8, 1947, now abandoned, for Methods of Performing RoutineLaboratory Analysis, which application will now be permitted to lapsewithout prejudice in view of its being superseded by this presentapplication.

At the present time, in the performance of many routine analyses,clinical laboratories make use of physical colorimetry andspectrophotometry as well as the practice of various related technics,such as those requiring the employment of photoelectric instruments andthose involving titrimetry. Analyses of this character have exceedinglybroad application and in the physiological field, for diagnosticpurposes, are extensively used in the analysis of body fluids such asblood serum and plasma, urine, and spinal fluid. Such procedures,although routine or research in nature, normally require painstaking andlaborious preliminary steps, including the exacting preparation ofquantitative standards for color comparison purposes and for checkingthe accuracy of the instruments utilized so that any deviations in theoperation thereof will be precisely adjusted for by calibration.customarily, whatever excess may remain of these standards after theaccomplishment of the color comparison or the instrument verification orcorrection procedure will usually be discarded since same are subject todeterioration, inadvertent dilution or contamination and, thus, theirfurther use at a later date will be productive of inaccuracy.Consequently, it is necessary to prepare such standards immediatelyprior to their use. This procedure is manifestly inherently uneconomicaland, furthermore, demands an element of proficiency on the part of theoperator in preparing the standard since any errors in the formationthereof will perforce cause the determination of false results.

Furthermore, the preparation of many of such standards are mostintricate and thereby demand marked skill and wide experience on thepart of the technician. It is evident that the amount of time spent byskilled personnel inpreparing such reagents constitutes a severe lack ofeconomy of such individuals time since it diverts them from theirimmediate research pursuits and thereby causes devotion of aconsiderable proportion of their time on merely ancillary preparatorysteps. With the developing shortage of highly trained scientificpersonnel, it is readily recognized that their efiorts should at alltimes be directed solely to the problem at hand and not dissipated onpreparatory procedures.

Therefore, it is a primary object of the present invention to provide amethod of preparing quantitative laboratory standards incorporating thepreparation of a dried, solid reagent of predetermined weight which maybe dissolved in a selected diluent to form a standard of predeterminedconcentration suitable for accurate quantitative measurement.

It is a further object of the present invention to provide a method forpreparing quantitative laboratory standards which comprises thedesiccation of a predetermined quantity of a solution of knownconcentration and the maintenance of such desiccated it will retain itsproperties indefinitely.

It is an additional object of the present invention to provide a methodfor preparing quantitative laboratory standards which may be accuratelyand reliably performed by relatively unskilled operators.

It is a still further object of the present invention to provide amethod of preparing quantitative laboratory standards Which is highlyeconomical, simple in performance, and convenient.

With the above and other objects in view, my

invention resides in the novel methods and proc esses presentlydescribed and pointed out in the claims.

In the accompanying drawing Figure 1 is a side elevational view of apreferred" form of reagent ampule for use in the performance of themethod of the present invention; Figure 2 is a vertical cross-sectionalview of the ampule shown in Figure 2; and

Figure 3 is a horizontal sectional view taken along line 3-3 of Figure1.

The method herein comprises th formation of a solution of desiredconcentration; the measuring of a predetermined quantity of the preparedsolution and the depositing of the same within an ampule or otherenclosable container; the dehydration or desiccation of the measuredquantity in the ampule by any suitable means, such as by evaporation orby lyophilization, as in the manner set forth in United State LettersPatent No. 2,225,774, issued to E. W. Flosdorf, December 24, 1940; thesealing of the ampule under vacuum; andthe ultimate utilization of theportion under conditions wherein wholly dried, solid reagent by ruptureof the ampule and dissolution of the dried reagent in a predeterminedquantity of selected diluent to reconstitute a quantitative standard ofknown concentration.

Referring now in more detail, and by reference characters to thedrawing, which illustrates a practical type of ampule for use in themethod herein taught, but is not a part of the present invention, Adesignates a blown or drawn glass ampule of essentially tubular form andintegrally comprising a lower or body portion 1, an intermediate orconstricted portion 2, and an upper portion 3 preferably ofsubstantially the same diametral size as the body portion l, tapering atits upper end into an axially upwardly projecting sealing neck 4, thelatter being initially open for filling purposes and, subsequent tofilling, being sealed, as at 5, in a hot flame, such as a Bunsen burneror blow' lamp. The constricted portion 2 is provided, midway of itslength, with a line of weakness or scratch line 5, and a small outwardlyblown or drawn nipplelik protuberance I, the apex or point of which iscoincident with the scratch line 6.

In the performance of the method of this invention, the reagent ampule Ais filled with an accurately measured or weighed quantity of reagenthaving a precisely determined concentration suitable for makinga reagentsolution of definite strength or normality when added to a statedquantity of distilled water or other diluent. The quantity of reagentthus deposited is dehydrated for reduction to a completely dried, solidstate. philization wherein the sealing neck 5 which is, as yet, stillopen, is connected by suitable rubber tubing toa high vacuum system (notshown), which may be of any conventional design or construction and thecontents of the ampule desiccated by freezing the liquid contents andconducting desiccation by sublimation. This method is particularlyadvantageous in dealing with various types of biological reagents whichmust be handled very carefully to avoid impairment of their action.After the product has been com pletely desiccated, the sealing neck 4 issealed ofi and the product. is completed, ready for labeling and finaluse. The precise conditions of such dehydration methods depend upon thecharacter of the particular standard being prepared as some will requirelow temperature desiccation whereas others are reduced to a dry state byhigh temperature vacuum procedures.

In some cases, such as with a bilirubin standard, lyophilization is notused but the dehydration process is carried out by evaporation procedurewith sealing of the desiccated material in an inert atmosphere, as willpresently be more fully described. The manner of desiccation is thusdependent upon the particular substances involved.

Reagents so ampulized in wholly desiccated state will keep forindefinite periods without deterioration, remaining stable as to theiroriginal properties, so that an operator may use same, as will be shownbelow, whenever the particular occasion arises without fear of anyinaccuracy resulting through untoward impairment of the reagent.

- When the operator desires to make a quantitative laboratory standardfor clinical analytical purposes, the ampule A is manually broken alongthe scratch lineB into two sections, and the contents-washed by a smallquantity of the indi- This may-be accomplished by lyocated diluent intothe container in which such standard is to be made. It is to beparticularly noted that exact quantitative transfer of the ampulecontents must be made. Therefore, the desiccated material in both of thesections of the now broken ampule must be completely dissolved andtransferred. The rupturing 0f the ampule A along the scratch line 8 willresult in the formation of a pouring lip to facilitate transference ofthe dissolved reagents into the container in which the standard is beingmade. Successive washings from a conventional laboratory wash bottle, orthe like may be done to assure that all desiccated material has beentotally dissolved and transferred. The solution is then made up to thepredetermined or stated volume.

The quantitative standards so prepared by the present invention areparticularly fitted for utilization in verifying the calibration tablesand graphs of photoelectric colorimeters and spectrophotometersas wellas providing color standards for visual colorimetry and for titrimetrictechnics. As illustrative of the method herein described, below arepresented two specific examples of the preparation of a quantitativestandard for checking the accuracy of the type of calibration tablesupplied with a pre-calibrated spectrophotometer or of atransmittance-concentration graph.

Example I The preparation of a hematin standard for use in the alkalinehematin technic for determining total hemoglobin is readily accomplishedby this method. Although hematin standards of various concentrationsmay, of course, be prepared, the example herewith relates to onerepresenting 18.7 grams per cent hemoglobin equivalent. A one litersolution is formed by dissolving 2.776 grams of hematin in a .2 molarborate bufier, pH 9.4. After incubation and aging of the reagent thusmade, 5 cc. thereof, containing a proportionate amount of hematin,namely 13.88 mg., is deposited into the ampule A through the now opensealing neck 4. The 5 cc. of reagent is then subjected to lyophilizationwhich comprises shell freezing by immersion of the ampule A into asuitable bath, such as isopropyl alcohol at 4 0 C. Subsequent to thefreezing step, and before thawing can occur, a high vacuum is applied inorder to evacuate .all water thereby leaving the reagent in a completelydesiccated, solid form. The ampule .A is then sealed under vacuum.

To reconstitute the reagent for analyticaluse, the ampule A is brokenalong the line of weakness 5, and smallamounts of distilled water, inthe neighborhood of 10 ml., are deposited in the upper and lowerportions I, 3, of the ampule A for dissolving the solid reagent. Thereonthe portions I, 3, are emptied into a 250 cc. flask, However, additionalflushings of the ampule A may be made to assure quantitative dissolutionof the entire reagent. The flask is then made up to the mark with thediluent, namely, .2 molar borate buffer pH 9.4, to constitute a standardof accurate known strength, suitable for use as a standard in visualcolorimetry and for use as a standard for calibration of verification ofphoto electric and spectrophotometric curves or calibration tables. As averification standard, it has a transmittance equivalent to 18.7 gramsper cent hemoglobin.

Example II U mining the total protein and albumin content of blood,plasma is extracted from whole beef blood and the protein contentthereof discovered by conventional technics. The beef plasma is thendiluted with a saline solution, namely, .85 per cent sodium chloride, inrequisite amount to provide 360 mg. of protein per 5 cc. Then 5 cc. ofthe reagent thus formed is deposited in the ampule A and subjected toshell freezing and desiccation, after which the ampule A is sealed undervacuum. The solid reagent Within the ampule A represents 2 grams percent total serum protein equivalent. To reconstitute the reagent, theampule A is broken and flushed repeatedly with small amounts of thediluent, which in this case is 30 per cent urea-thymol reagent, untilthe reagent is completely quantitatively dissolved, with transferencebeing made to a 100 ml. volumetric flask. The diluent is then added tothe flask to make up to the mark. Various concentrations of proteinstandard reagents may be made by this method, such as, one containing280 mg. per 109 ml. which represents 7 grams per cent total proteinmaterial or one of the concentration of 160 mg. per 100 ml., whichrepresents 4 grams per cent total serum protein equivalent, and beingsuitable for use in colorimetry and for calibration for verification ofphotoelectric and spectr0pho tometric curves or calibration tables.

Thus, by standards of the type a technician may accordingly verify orcorrect calibration tables to assure accurate quantitative measurement.

The preparation of a bilirubin standard for use in quantitativelydetermining the presence of this t bile pigment in serum or plasma, willserve as an example of the preparation of a reagent by evaporation. Inthis instance, a solution is made by dissolving 20 mg. bilirubin in 100cc. of chloroform. Then 5 cc. of the reagent thus formed is deposited inthe ampule A and subjected to evaporation to provide 1 mg. of bilirubinin a dry, solid state, after which the ampule A is sealed in an inertatmosphere, with nitrogen being the gas used. Thus, the air in theampule is replaced by nitrogen, the presence of which will preventoxidation of the bilirubin to biliverdin. The reagent is reconstitutedfor chemical and clinical analytical use by breaking the ampule A anddissolving completely the desiccated bilirubin with small amounts ofchloroform, and then quantitatively transferring the dissolved bilirubinby methyl alcohol to a 100 cc. volumetric flask. Methyl alcohol is thenadded to the flask to make up to the mark. As described hereinabove, itis understood that both sections of the broken ampule may be repeatedlywashed to assure exact and. precise dissolution of all bilirubin. Thetotal amount of diluent utilized is roughly 95 per cent methyl alcoholand 5 per cent by volume chloroform. The solution so formed represents 1mg. per cent bilirubin equivalent and will accordingly provide accuratelight transmittance for such concentration to serve as a reliableworking standard.

It should be understood that the present invention resides in the uniquemethod of providing dry, solid reagents of known strength from asolution of predetermined concentration, which reagent is adapted forreconstituting a solution of the identical concentration at a subsequenttime for quantitative measurement purposes in chemical and clinicalanalysis.

It is, of course, apparent that the present method is equally designedfor use by physicians,

above set forth,

analytical chemists, and laboratory technicians in both the medical andindustrial fields, and obviously does not ing an incorrect standard. Theeconomy in material, time, and labor implicit in the practice of themethod is apparent.

It should be understood that changes and modifications in the methodsabove set forth and in the various steps of their production may be madeand substituted for those herein shown and described without departingfrom the nature and principle of the present invention.

Having thus described my invention, What I claim and desire to secure bLetters Patent is:

1. The method of making a hematin standardized reagent for use incalorimetry and in checking the accuracy of spectrophotometriccalibration tables which method comprises placing an accurately measuredquantity of hematin in a measured quantity of borate buffer solution toprovide a liquid reagent of predetermined concentration, placing anaccurately measured quantity of the so-formed liquid reagent within anenclosable container, freezing the liquid reagent within the enclosablecontainer, subjecting the frozen material to a high vacuum system forcomplete desiccation of the reagent to provide a solid standardizedreagent, then closing the container, opening the container at the timeof preparation of the standard, and depositing the entire contents ofthe container into an accurately measured and predetermined quantity ofa borate bufier solution in order to reconstitute the liquid reagent inits original strength.

2. The method of making a hematin standardized reagent for use incolorimetry and in checking the accuracy of spectrophotometriccalibration tables which method comprises dissolving an accuratelymeasured quantity of crystalline hematin in a measured quantity ofborate buifer solution having pH 9.4 to provide a liquid reagent ofpredetermined concentration, depositing an accurately measured quantityof the so-formed liquid reagent within an enclosable container, freezingthe liquid reagent within the enclosable container, subjecting thefrozen material to a high vacuum system for complete desiccation of thereagent to provide a solid standardized reagent, then closing thecontainer, opening the container at the time of preparation of thestandard, dissolving the solid reagent in a small amount of distilledwater, and then depositing the dissolved reagent into an accuratelymeasured and predetermined quantity of borate buiier solution in orderto reconstitute the liquid reagent in its original strength.

THEODORE E. WEICHSELBAUM.

References Cited in the file of this patent UNITED STATES PATENTS OTHERREFERENCES How to Make Standard Volumetric Solutions in a Few Minutes,E. H. Sargent & Co., Chicago, 111., 4 pg. pub.

1. THE METHOD OF MAKING A HEMATIN STANDARDIZED REAGENT FOR USE INCOLORIMETRY AND IN CHECKING THE ACCURACY OF SPECTROPHOTOMETRICCALIBRATION TABLES WHICH METHOD COMPRISES PLACING AN ACCURATELY MEASUREDQUANTITY OF HEMATIN IN A MEASURED QUANTITY OF BORATE BUFFER SOLUTION TOPROVIDE A LIQUID REAGENT OF PREDETERMINED CONCENTRATION, PLACING ANACCURATELY MEASURED QUANTITY OF THE SO-FORMED LIQUID REAGENT WITHIN ANENCLOSABLE CONTAINER, FREEZING THE LIQUID REAGENT WITHIN THE ENCLOSABLECONTAINER, SUBJECTING THE FROZEN MATERIAL TO A HIGH VACUUM SYSTEM FORCOMPLETE DESICCATION OF THE REAGENT TO PROVIDE A SOLID STARDARDIZEDREAGENT, THEN CLOSING THE CONTAINER, OPENING THE CONTAINER AT THE TIMEOF PREPARATION OF THE STANDARD, AND DEPOSITING THE ENTIRE CONTENTS OFTHE CONTAINER INTO AN ACCURATELY MEASURED AND PREDETERMINED QUANTITY OFA BORATE BUFFER SOLUTION IN ORDER TO RECONSTITUTE THE LIQUID REAGENT INITS ORIGINAL STRENGTH.